1. Field of the Invention
This invention covers the field of medicine and can be used for obtaining of major constituents of connective tissues including sulphated glycosaminoglycans (sGAG) for application as pharmaceutical substances in medicine and veterinary medicine, drugs and preventive materials, e.g. eye drops, ointments, suspensions etc. or in the composition of medical products.
2. Description of the Related Art
A method of obtaining sGAG from the trachea of cattle and shark's fins including the hydrolysis of an acetone powder suspension from tissues with 1% papain solution for 24 hours at 62° C., the subsequent precipitation of hydrolyzate with acetone, sediment dissolution in saline, decoloration with use of KMnO4 and precipitation with acetone (DE 2037942, A61K31/725) is known.
However, this method is simple and convenient enough, but it does not obtain a high-purity sGAG preparation. Moreover, this method does not ensure high recovery of sGAG and its realization is very expensive.
A method of extraction of sulphated glycosaminoglycans from the cornea including the treatment of the cornea of farm livestock with water solutions and ethanol with the subsequent boiling (RU 2056851) is known.
The disadvantage of this method is a partial loss of glycosaminoglycans, such that a part of the glycosaminoglycans remains in the sediment by dialysis, in particular keratan sulfate. Furthermore, a part of the glycosaminoglycans remains in the non-destroyed tissue by enzymatic treatment of the cornea, which leads to the reduction of end product output, and only solute sulphated glycosaminoglycans can be obtained with use of this method.
A method of extraction of glycosaminoglycans from fibrous cartilage is known, by which a tissue sample is exposed to enzyme proteolysis, specific components of glycosaminoglycans are extracted from the obtained hydrolyzate by precipitation of them from the hydrolyzate by quaternary ammonium bases or detergents and their stepwise purification and quantification is carried out (Adams N. E. Muir H. The glycosaminoglycans of canine menisci/Biochem. J. 1981. Vol. 197, No. 2. P. 385-389).
A method of extraction of glycosaminoglycans from tissues forming elements of a joint including the fibrous cartilage is also known, which consists in enzyme proteolysis of a minced tissue sample in 0.1 M acetate buffer containing papain in a quantity of one fourth of fresh weight at the temperature of +60±4° C. for 24 hours, deproteinization of the hydrolyzate obtained by the trichloroacetic acid for 24 hours at the temperature of +4° C., purification of extracted glycosaminoglycans with the help of their precipitation with ethanol containing calcium acetates or sodium acetates for 24 hours at the temperature of +4° C., and quantification of glycosaminoglycans (E. V. Karyakina. D. V. Kosyagin. Features of extraction of glycosaminoglycans from tissues forming elements of a joint/The All-Union Rheumatologists Conference: Lecture theses (Dec. 7-9, 1988). Moscow, 1988, P. 108-109).
The disadvantage of the known methods is a long duration of study (4-5 days), which does not allow the obtaining of current information on the state of intra-articular structures of connective tissue.
A method of obtaining of sulphated glycosaminoglycans is known, which consists in the treatment of the cornea of farm livestock with water solutions and ethanol with the subsequent boiling, in that the corneas of farm livestock are put into the half-and-half mixture of saline and distilled water with the ratio of the cornea and water mixture 1:1, frozen repeatedly three times at 0° C., reduced to fine particles of 0.1×0.1 mm, then ethanol is added in concentration of 1%, the mixture is homogenized, ethanol is added repeatedly to the final concentration of 5%, and then the mixture is boiled for 5-10 minutes. (RU 2118524, A61F9/00 A61K35/14 publ. Sep. 10, 1998).
The disadvantage of the mentioned method consists in the laborious expensive treatment, which does not provide for full output and liberating of sGAG from the solution, and also in impurities of protein and other antigenic molecules being present in the mentioned tissues, which sufficiently reduces the purity of the end product.
This method of obtaining sGAG from the cornea is selected as a prototype, as it the most closely relates to the invention proposed with respect to its technical decision.